S. aureus is an opportunistic bacterial pathogen responsible for a diverse spectrum of human and animal diseases. Approximately 90 percent of S. aureus strains are encapsulated. Although 11 capsular serotypes have been described, types 5 and 8 constitute 75 percent of clinical isolates. Type 5 and 8 polysaccharides are structurally very similar to each other; they differ only in the linkages between the sugars and in the sites of O- acetylation. The overall goal of this study is to use a molecular approach to define the genetic organization and functions of type 5 capsule genes in S. aureus. The molecular events controlling the biosynthesis, transport, and assembly of the S. aureus capsular polysaccharide will be examined, and the relation of the type 5 genes to those from other capsular serotypes will be investigated. Specific Aims include determining the nucleotide sequences of DNA fragments that complement capsule expression in mutant strains. Computer analysis will be used to identify open reading frames, the predicted amino acid sequence, and homology with known gene sequences. Based on the predicted pathway for capsule biosynthesis in S. aureus, the appropriate biosynthetic enzyme activities will be assessed from products of the cloned genes in Escherichia coli and S. aureus. Reduced enzyme activity in mutant S. aureus strains will also be assessed. Type 5 capsule genes identified as a result of this study will be located on the physical map of the S. aureus chromosome by the combined techniques of pulsed field gel electrophoresis and DNA hybridization. The genetic organization of the type 5 gone region in S. aureus will be further investigated. To do this, additional type 5 mutants will be generated using transposon Tn917-LTV 1. Mutants will be screened for defects in capsule expression by colony immunoblots using specific antibodies. Mutations affecting capsule expression will be transferred back to the wild-type strain to insure that the transposon insertion is responsible for the effect on capsule expression. Tn917-LTV 1 insertions will be mapped to cosmid clones carrying the capsule gene region, and the specific defects in capsule expression will be determined by immunologic and chemical assays. To understand the genetic regulation of capsule expression in S. aureus, the influence of the known regulatory loci agr, sar, and xpr on type 5 capsule expression will be determined. Each of the regulatory genes, insertionally interrupted by a transposon, will be transferred to the type 5 strain Reynolds by transduction or transformation. Recombinants will be assessed for phenotypic changes associated with the regulation defect and for capsule expression. These studies should lead to a better understanding of the organization, structure, function, and regulation of capsule type 5 expression in S. aureus.